The present exemplary embodiments relate to the imaging arts and find particular application in conjunction with low and high-density cell detection, locating, and identifying in blood smears, biological assays, and the like across distinct imaging systems, and will be described with particular reference thereto. However, it is to be appreciated the exemplary embodiments will also find application in imaging, locating and identifying other types of low- or high-density features on various substantially planar surfaces and samples, such as imaging semiconductor wafers, imaging particulate contaminants in fluids or thin solid films, and so forth, with such imaging finding specific uses in the printing arts, electronic arts, medical arts, and other scientific and engineering areas.
In rare cell studies, a particular problem arises due to the typically low concentration of the rare cells in the blood or other body fluid. In a typical rare cell study, blood is processed to remove cells that that are not needed. Then a probe, such as a fluorescent material, is applied that attaches to antibodies, which in turn selectively attach to a cell surface or cellular protein of the rare cells. The cellular proteins may be membrane proteins or proteins within a cell, such as cytoplasm proteins. The antibodies may also attach to other types of molecules of the rare cell, as well as to DNA.
The fluorescent material may be a fluorescent marker dye or any other suitable material which will identify the cells of interest. A smear treated in this manner, which may include the blood and/or components of the blood, is prepared and optically analyzed to identify rare cells of the targeted type. For statistical accuracy it is important to obtain as large a number of cells as required for a particular process, in some studies at least ten rare cells should be identified, requiring a sampling of at least ten million cells, for a one-in-one-million rare cell concentration. Such a blood smear typically occupies an area of about 100 cm2. It is to be understood, however, that this is simply one example and other numbers of cells may be required for statistical accuracy for a particular test or study. Other cell identifiers which are being used and investigated are quantum dots and nano-particle probes. Also, while a rare cell is mentioned as a one-in-one-million cell concentration, this is not intended to be limiting and is only given as an example of the rarity of the cells being sought. The concepts discussed herein are to be understood to be useful in higher or lower levels of cell concentration.
The ability to scan large numbers of cells at a high rate is considered a key aspect which increases the throughput of testing processes. Therefore, it is considered valuable to provide a system which improves the speed, reliability and processing costs which may be achieved by cell detection systems and/or processes.
Fiber array scanning technology (FAST) provide particular systems and processes that increase the speed at which scanning of a sample and the detection of potential or candidate rare cells may be accomplished, and therefore lends itself to the investigation of large samples.
In general, in FAST systems, a sample of material to be examined is treated with fluorescent material (e.g., a selectively attaching or labeling dye), typically by introducing a measured amount of the material into the sample itself. The sample is applied to a slide, and radiation, typically laser light, is scanned across the sample. This provides essentially a raster scanning of the sample, with the position of incidence of the laser on the sample, usually relative to a fiducial mark on the slide, precisely known at all times. The output intensity of the light resulting from the scanned laser beam varies with position on the surface of the sample. Appropriate hardware detects selected wavelengths of the output light, and a processor analyzes the detected output light and associates fluorescence events and locations.
In FAST systems, and most other detection systems, it is important to be able to rule out as many false positives (i.e., incorrect identification of a target cell) as possible. One source of such false positives is dust, debris, healthy cells, etc., which emit light in response to the incident input light even without the attachment of fluorescent dye (referred to herein as auto-fluorescence). In order to identify auto-fluorescence events, and thus enable ruling out such false positives, when a single dye is employed, it is typical to use a large enough quantity of dye in the sample so fluorescence events are much brighter than the auto-fluorescence events. However, the dye used is relatively expensive, and when too much dye is used, it loses its ability to selectively attach to the target cells, instead saturating the sample and attaching to a variety of unintended components in the sample.
In another approach to reducing false positives due to auto-fluorescence, two different dye materials are introduced into the sample, each dye material fluorescing at a different wavelength in response to incident radiation. Each dye attaches relatively equally to the target cells. Since auto-fluorescence events are brightest closer to the input wavelength, one of the dyes is chosen such that its fluorescence wavelength is relatively far from the source wavelength. Fluorescence events are examined at the output wavelengths of both dyes. Fluorescence events predominantly at only one of the wavelengths are considered to be false positives, such as would arise form auto-fluorescence, while events emitting at both wavelengths are considered to be indicative of the presence of a target cell.
This dual-dye arrangement has the disadvantage that significant amounts of dye need to be introduced into the sample. In addition, in certain instances, the ratio of one “color” of dye to the other must be carefully controlled. For example, when scanning with a 488 nm laser and sampling with “green” and “red” dyes (i.e., dyes that fluoresce in the green and red wavelength bands, respectively), the light emitted due to auto-fluorescence is greater in the green channel than in the red channel. This requires balancing the ratio of the two dyes such that fluorescent emission caused by the incident laser light is stronger in the red than in the green portions of the spectrum. Excitation is significantly less efficient for the red dye than for the green dye. Thus, significantly more red dye must be introduced into the sample. Ratios of 33:1 red to green are typical. The aforementioned cost and saturation problems, as well as cross-talk and interference between the fluorescing, can therefore arise.
To overcome issues related to the use of two different dyes, U.S. Ser. No. 11/018,759 provides a technique for alternative scanning of two separate laser beams in an attempt to minimize cross-talk and interference between the fluorescence which occurs through the use of two different probes emitting at different wavelengths. However, this technique has its own drawbacks, including the previously mentioned expense and complication of sample preparation and data analysis. Particularly, and as previously mentioned, because the excitation of a first probe, for example, a red dye may be less efficient than a second probe, for example, a green dye, about 33 times more red dye must be used than green dye. This requirement makes the processing of samples more complex and increases overall expense. Further, the use of high amounts of red dye or other probe materials may, again, cause accumulations or aggregation of the dye or materials, which is undesirable.
Therefore, there is a need in the art for a process and system for scanning for rare cells using a single laser with reduced cost and complexity, such as requiring only a single dye, yet is capable of ruling out false positives such as those occurring due to auto-fluorescence events.